The present invention relates to sensing or determining the concentration of an analyte of interest in a medium. More particularly, the invention relates to sensor apparatus or systems and methods for sensing the concentration of an analyte of interest, for example, oxygen, in a medium, for example, blood. It is sometimes necessary or desirable for a physician to determine the concentration of certain gases, e.g., oxygen and carbon dioxide, in blood. This can be accomplished utilizing an optical sensor which contains an optical indicator responsive to the component or analyte of interest. The optical sensor is exposed to the blood, and excitation light is provided to the sensor so that the optical indicator can provide an optical signal indicative of a characteristic of the analyte of interest. For example, the optical indicator may fluoresce and provide a fluorescent optical signal as described in Lubbers et al. U.S. Pat. No. Re 31,897 or it may function on the principles of light absorbance as described, for example, in Fostick U.S. Pat. No. 4,041,932.
The use of optical fibers has been suggested as part of such sensor systems. The optical indicator is placed at the end of an optical fiber which is placed in the medium to be analyzed. This approach has many advantages, particularly when it is desired to determine a concentration of analyte in a medium inside a patient's body. The optical fiber/indicator combination can be made sufficiently small in size to easily enter and remain in the cardiovascular system of the patient. Consistent and accurate concentration determinations are obtained. Luminescence measurement analysis for monitoring concentrations of analytes is well known in the art. Generally, a calibration curve of light intensity (or a function of intensity) vs. concentration of the analyte is made. This method may involve a determination of absolute light intensities of both excitation and emission. In phase modulation detection methods, luminescent indicators are excited by an intensity modulated excitation source and the phase shift between the excitation and emission signals can be used to determine an analyte dependent luminescence lifetime.
One problem which may exist in such systems is the wavelength proximity between the excitation signal (light) and the emission signal (light) of the indicator. In many cases, the excitation signal and emission signal each have relatively similar wavelengths. This can result in misinterpreting the emission signal, which misinterpretation results in an inaccurate determination of the analyte concentration. It would be advantageous to provide a sensing system in which the wavelengths of the excitation and emission signals are substantially different.
It would be advantageous to provide a sensor system in which the lifetime, and preferably both the lifetime and intensity, of the differentiated emitted signal are sensitive to analyte concentration in a medium. Emissions, for example, fluorescent emissions, which are sensitive or dependent in terms of both lifetime and intensity to analyte concentration are said to be dynamically quenchable by the analyte.
While substantial differentiation of the excitation and emission signals can be achieved using phosphorescent organic indicators, dye stability is often a problem. Indeed, Stanley et al. U.S. Pat. No. 3,725,648, explicitly excludes phosphorescent molecules as potential sensor materials because of the stability problems. Some phosphorescent platinum group inorganic complexes and phosphorescent lanthanide complexes have shown suitable stability but are quite expensive and may generate significant amounts of highly reactive singlet oxygen upon irradiation in the presence of oxygen. It would be advantageous to provide a sensing system in which substantial differentiation of excitation and emission signals is achieved using stable dyes, e.g., with reduced, or no, generation of reactive species, such as singlet oxygen.
Substantial differentiation of the excitation and emission signals can be achieved with an excited singlet state by using energy transfer from an analyte responsive first dye to an analyte insensitive second dye (Barner et al. European Patent No. 381,026). Energy transfer is a collisionless process that occurs through space and requires substantial optical overlap between the characteristic emission of the first dye and the characteristic absorption of the second dye. The first dye absorbs light at a characteristic excitation wavelength and its excited state energy is transferred to the second dye. In this way, the second dye can emit at its own characteristic emission wavelength in response to excitation of the first dye. An analyte that quenches the emission from the first dye also quenches the energy transfer to the second dye. Therefore, the emission of the second dye is indirectly influenced by analyte and a substantial shift between excitation and emission wavelengths is achieved. For energy transfer to occur several requirements must be met. First, two different indicators are required and both indicators must fluoresce. Second, there must be overlap between the characteristic emission of the first dye and the characteristic absorption of the second dye. Third, to prevent analyte insensitive emission, the second dye must not absorb light at the excitation wavelength of the first dye. In addition to the above requirements, excited state collisional interactions between dye molecules can quench energy transfer. Barner et at. discloses methods to prevent unwanted collisional interactions.
Unwanted collisional interactions are further discussed by Sharma et al. in "Unusually Efficient Quenching of the Fluorescence of An Energy Transfer-Based Optical Sensor for Oxygen", Analytica Chimica Acta, 212 (1988) 261-265. They disclose a two fluorophor energy transfer based sensor consisting of pyrene (their analyte responsive first dye) and perylene (their analyte insensitive second dye) both dispersed in silicone rubber. In response to excitation of the pyrene using a 320 nm excitation signal a fluorescent signal (characteristic of the emission wavelength of perylene) is observed at 474 nm. Sharma et al. concluded that the 474 nm emitted signal resulted in part from perylene emission and in part from a collisional interaction between the pyrene and perylene thereby forming an emissive excited state complex which they called an exciplex. The interference of exciplex emission with perylene emission is presently believed to be undesirable. No covalent bonding of pyrene and perylene to the silicone rubber is disclosed so that there is not teaching or suggestion as to how such covalent bonding affects the system. It would clearly be advantageous to employ an emitted signal for analyte concentration determination that is substantially removed or resolved from other emissions in the system. Furthermore, it would be advantageous to obtain this signal by some means other than energy transfer.
Pyrene excimer emission has been successfully employed in solutions for the detection of organic compounds (Ueno et al., Anal. Chem., 1990, 62, 2461-66) and anesthetics (Merlo et al., IEEE Engineering in Medicine & Biology, 11th Annual International Conference Proceedings, 1989), with excimer fluorescence serving as a reporter of the aggregation of cyclodextrins or of a change in the local viscosity, respectively. These sensing mechanisms work on the premise of an analyte suppressing or enhancing the efficiency for population of an emissive excited state. These systems are not disclosed as being usable in solid sensing elements. An additional problem with such systems is that they are incompatible with phase modulated detection methods, since the lifetime of the emissive state is substantially independent of analyte concentration.
Fiber-optic based sensors are very useful, for example, in medical applications. One problem which may exist with such systems is related to the inherent flexibility of optical fibers. These flexible fibers have a tendency to bend which, in turn, distorts the signals being transmitted by the fibers to the signal processor. Signal distortion caused by fiber bends or other sensor system problems result in inaccurate concentration determinations. It would be advantageous to provide a sensor and concentration determination method which provide accurate concentration data in spite of such distortions.
Seitz et al. (U.S. Pat. 4,548,907) discloses a fluorescence-based optical sensor which includes a fluorophor having an acid form and a base form. Specifically, the relative amounts of the acid form and base form vary depending on the pH of the medium. The fluorophor is excited at two different wavelengths, one for the acid form and one for the base form, and fluorescence signals at a single wavelength are detected. By ratioing the fluorescence signals obtained at the two different excitation wavelengths, the pH of the medium can be determined independent of amplitude distortions which affect both signals equally. This sensor has the advantage of using a single fluorophor. However, the sensor of this patent is limited in that only those analytes which influence the ratio of acid form to base form of the fluorophor can be monitored. Again, the emission lifetimes are substantially independent of analyte concentration. Also, no other multiple state optical indicators are taught or suggested.
Lee, et al. in "Luminescence Ratio Indicators for Oxygen", Anal. Chem., 59, p. 279-283, 1987, report on work the goal of which was to develop a single reagent that would show two luminescence bands, a shorter wavelength "analytical" band subject to quenching by oxygen and a longer wavelength "reference" band independent of oxygen levels. Specifically, the work was to formulate a system showing both shorter wavelength oxygen-sensitive pyrene monomer emission and longer wavelength oxygen-insensitive pyrene dimer emission. This work did not succeed in finding a ratio-based indicator system to measure oxygen in aqueous systems. Further, as noted above, using a shorter wavelength oxygen sensitive emission can result in oxygen concentration determination inaccuracies because of possible overlapping between this short wavelength emission and the excitation signal.
Canadian Patent Application 2,015,415 (Divers et al.) discloses an oxygen sensor including a single species of indicator selected from perylene derivatives dispersed or immobilized in a crosslinked polydimethylsiloxane matrix which gives a shorter wavelength oxygen sensitive emission and a longer wavelength oxygen insensitive emission and, thus, can be used as both the indicator and the reference element. Using shorter wavelength oxygen sensitive emission can result in inaccuracies because of overlap with the excitation signal, as described above. Also, there is no teaching or suggestion that the shorter and longer wavelength emissions are the result of different forms of the indicator. To the contrary, the document implies that a single indicator species provides an oxygen-sensitive emission region and a different oxygen-insensitive emission region.
European Patent Publication 0 363 219 (Barnes) discloses an oxygen sensing apparatus using Europium or Erythrosin-B as phosphors which are excited with a mono-chromatic light that is sine wave modulated in the kHz regime. The emitted light of a different wavelength is also sine wave modulated, with the phase difference between the two sine waves being a measure of the quenching effect of oxygen and, thus, a measure of the partial pressure of oxygen. This publication does not disclose the use of any other indicators, for example, fluorescent indicators. Modulation in the kHz region cannot be extended to shorter lived fluorescent indicators because the phase offsets introduced by transmission of the excitation and emission signals, e.g., through an optical fiber, become significant. Also, there is no teaching or suggestion that the phosphors used produce different emitting forms. Further, as noted above, the use of phosphorescent indicators can result in other problems.